Soil Silicon Amendment Increases Phyllostachys Praecox Cold Tolerance in a Pot Experiment

Article Soil Silicon Amendment Increases Phyllostachys praecox Cold Tolerance in a Pot Experiment

Zhuang Zhuang Qian 1,2, Shun Yao Zhuang 1,*, Qiang Li 1 and Ren Yi Gui 3

1 State Key Lab of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008, China; [email protected] (Z.Z.Q.); [email protected] (Q.L.) 2 College of Forestry, Nanjing Forestry University, Nanjing 210037, China 3 State Key Lab of Sub-Tropical Sivlculture, Zhejiang A & F University, Hangzhou 311300, China; [email protected] * Correspondence: [email protected]

Received: 15 March 2019; Accepted: 7 May 2019; Published: 10 May 2019

Abstract: Cultivated bamboos are occasionally subjected to cold stress in winter, and silicon could improve their cold tolerance. However, evidence of the effect of Si on bamboos is still limited. Therefore, a batch and pot experiment was conducted for six months to investigate the effects of 1 different Si fertilizer application rates (0, 0.5, 1.0, 2.0, 4.0, and 8.0 g kg− of soil weight) on the physiological responses and photosynthesis parameters of Phyllostachys praecox under a simulated cold stress condition. The cold temperature was set to 5 ◦C, 0 ◦C, and 5 ◦C, successively. The bamboo − 1 biomass increased significantly when the Si amendment rate was at least 2.0 g kg− (P = 0.002), 1 and the highest biomass increase and root-to-canopy ratio were obtained with the 4.0 g kg− Si amendment. Furthermore, the Si contents in all organs of the bamboos increased with the increase of the Si amendment rate. The highest content of Si among the other organs was observed in the 1 1 leaf, and the content was 68.95 mg kg− with the treatment of 4.0 g kg− . With the application of Si, the photosynthesis rate of bamboo leaves was significantly increased (P = 0.008). The Si-amended bamboo exhibited a cold tolerance that was associated with stimulating antioxidant systems, and the enzyme activities of superoxide dismutase, peroxidase, and catalase increased with the increase of the Si amendment rate, whereas the malondialdehyde content and cell membrane permeability decreased with all Si treatments. A low temperature of 5 C exerted effects on the bamboo leaf chloroplasts, − ◦ but the ultrastructures of the chloroplasts remained intact after Si treatment. These findings suggest that Si fertilizer enhances bamboo growth and the tolerance of bamboo plants to cold stress. However, 1 a high application rate (8.0 g kg− ) caused a decline in the bamboo biomass, compared to T4. Thus, 1 a Si fertilization rate of 2.0~8.0 g kg− is recommended for bamboos under cold conditions.

Keywords: bamboo forest; cold stress; physiological response; silicon fertilization

1. Introduction Silicon is not a necessary element for higher plants, but it is essential for obtaining a high and sustainable yield for Poaceae crops. Si promotes the growth of various plants, especially those under abiotic and biotic stress conditions [1]. It is eﬃcient in alleviating abiotic stresses, including salt stress, metal toxicity, drought stress, radiation damage, nutrient imbalance, high temperature, and freezing [2–9]. Previous studies have suggested that the following possible mechanisms underlie the Si-enhanced resistance of plants to abiotic stress: Stimulating antioxidant systems [10–13], reducing the transpirational bypass ﬂow [14], reducing malondialdehyde (MDA), and improving root traits and the photosynthetic rate [15].

Forests 2019, 10, 405; doi:10.3390/f10050405 www.mdpi.com/journal/forests Forests 2019, 10, 405 2 of 13

Cold stress is an abiotic stress that causes severe damage to membranes [16], and an increase in reactive oxygen species (ROS) in plants caused by freezing as well as increased lipid peroxidation, arising from the accumulation of ROS, are the major causes of membrane damage. MDA is a harmful lipid peroxidation product, which could reflect the extent of oxidative damage [17]. Under an ROS burst, superoxide dismutase (SOD) constitutes the first line of defense against ROS [18]. The activity of SOD has marked positive effects on the antioxidant capacity of plants. Peroxidase (POD) is generally considered to be a merely ROS-detoxifying enzyme [19]. Catalase (CAT) is a specific enzyme that catalyzes the dismutation of H2O2 into O2 and H2O, preventing the damaging effects of H2O2 accumulation and protecting cells from oxidative stress [20]. Silicon has been shown to ameliorate the damage of cold stress on plants. Liu et al. [21] found that Si addition increased antioxidant activities and decreased the MDA of Cucumis sativus cv. under chilling stress. Zhan et al. [22] also showed that Si amendment alleviates chilling stress in D. brandisii plantlets, exhibiting increased CAT and SOD activities and decreased MDA. Moreover, Si forms deposits and undergoes polymerization, forming “phytoliths” in cells and intercellular spaces [23,24]. The strength and rigidity of the tissues improves [25], and thus cold tolerance is enhanced. Several researchers concluded that Si not only acts as a physical or mechanical barrier in plants but is also involved in metabolic and physiological activities [26]. Despite the wealth of information on the beneficial effects of Si on plants, the mechanisms underlying Si-mediated alleviation under freeze stress remain poorly understood. Phyllostachys praecox is among the bamboos with edible shoots in the region of Southeast China. This bamboo species has a high yield and plays an important role in the local economy. Accordingly, its sustainable production is essential for farmers. However, it is subjected to low temperatures and freeze stress in winter and often shows poor growth. Zhou et al. [27] reported that the ice damage rate of typical subtropical forests varied between 25% and 81%. A previous study found that the shoot output decreased by 32% per hectare in 2008 [28], and the bamboo freeze injury index of Lin-an City was 51% in 2016 [29]. Thus, measures to mitigate low-temperature stress are beneficial for bamboo production. Graminaceous plants absorb much more Si than other species [30]. Similar to many Poaceae species, bamboos accumulate Si, which may alleviate stress at low temperatures and freeze stress. However, Si uptake and accumulation in P. praecox, and the effect of Si on the resistance of P. praecox to low temperatures, have not been studied. Therefore, this study aimed to (1) investigate how Si amendment to P. praecox affects Si accumulation, (2) elucidate the relationship between Si content and P. praecox growth, and (3) explore the mechanism underlying the enhanced resistance of plants to low temperatures, after Si addition, by performing a plant physiological indicator analysis.

2. Materials and Methods

2.1. Bamboo and Soil

P. praecox is a bamboo that is mainly cultivated in Lin-an City (30◦16024.58” N, 119◦35014.59” E), with intensive management. Bamboos usually grow from seeds, underground rhizomes, or P. praecox flowers in the study site, but they do not form seeds, and they extend through the underground rhizome. Therefore, we selected bamboo rhizome for our pot experiment. The length of rhizomes was approximately 200 mm, their diameter was approximately 10 mm, and their weight was approximately 300 g. The rhizome sprouts of P. praecox were excavated in 2015 from the bamboo field and cultured in a greenhouse. Before the pot experiment, rhizome sprouts with similar sizes and weights were incubated. Soil (0–25 cm) in the pot experiment was collected from the bamboo garden in Zhejiang Agriculture and Forestry, Lin-an City, ZheJiang Province, China. The soil type was classified as Ultisol. The soil 1 basic physicochemical properties were as follows: pH, 5.31; available Si, 43.51 mg kg− ; soil organic 1 1 1 matter, 16.8 g kg− ; soil total nitrogen, 769 mg kg− ; available potassium, 25 mg kg− ; hydrolyzed 1 1 nitrogen, 81.62 mg kg− ; and available phosphorus, 68.5 mg kg− . The soil sample was air-dried, passed through a 2 mm mesh sieve, and, for the improvement of the soil structure, sieved soils were mixed with perlite (bamboo rhizomes prefer soil that is loose and permeable and are easily damaged Forests 2019, 10, 405 3 of 13 under hypoxia conditions, so perlite can increase the permeability of soil, which is beneficial for the growth of bamboo) in a 3:1 ratio (volume volume-1) for the pot experiment.

2.2. Bamboo Pot Experiment The bamboo pot experiment was conducted in 2016 in the greenhouse in Zhejiang Agriculture and Forestry, China. The light transmittance rate in the greenhouse was 88%, and the temperature ranged from 20 to 28 ◦C, which was suitable for bamboo growth. The height of the experimental plastic pots was 250 mm, and the diameter of the pots was 300 mm. Each pot had five holes for drainage in the bottom and was filled with 3 kg of soil. The experiment involved six treatments, 1 with Si fertilizer application rates of 0, 0.5, 1.0, 2.0, 4.0, or 8.0 g kg− , marked as T0, T0.5, T1, T2, T4, and T8, respectively. Each treatment had 20 pots, and four bamboos were planted in each pot. The photosynthesis parameters (photosynthesis rate, water use efficiency, CO2 of intercellular space, and stomatal conductance) of the bamboo leaves were measured every month. After six months, five bamboo pots from each treatment were randomly selected for biomass measurement. The root, rhizome, leaf, and stem of the bamboo plant were collected for further analysis. The root-to-canopy ratio was calculated by the underground biomass and overground biomass, and the equation was:

underground biomass root to canopy ratio = . (1) − − overground biomass

Low Temperature Incubation Three bamboo pots (bamboos in pots were basic, with the same height and ground diameter) were chosen randomly from every treatment for further incubation. The three bamboo pots from each treatment were divided into three culture boxes, and the temperature of the three culture boxes was set to 5, 0, and 5 C. A total of 18 bamboo pots were cultured for three days under the speciﬁed − ◦ temperatures. After the three types of temperature treatments (5, 0 and 5 C), the functional leaves of − ◦ bamboo were collected for plant physiological analysis.

2.3. Si in Plant Measurement The excavated bamboo was washed and divided into root, leaf, stem, and rhizome. It was then soaked in 0.5 M HCl for 20 s, followed by three to four rinses in distilled water, and then dried to stable weight at 65 ◦C. After being dried, the bamboo samples were ground and passed through a 0.25 mm mesh sieve for Si measurement. The samples were then microwave digested in a mixture 1 1 1 of 3 mL of 62% (w w− ) HNO3, 3 mL of 30% (w w− ) hydrogen peroxide, and 2 mL of 46% (w w− ) 1 hydroﬂuoric acid (HF), and the digested sample was diluted to 100 mL with 4% (w v− ) boric acid. The Si concentration in the digested solution was determined by the colorimetric molybdenum blue method at 600 nm [31].

2.4. Photosynthesis Parameter Measurement and Physiological Indicator Analysis For the photosynthesis parameter measurement, three sunlight-exposed leaves of each bamboo plant were measured, two bamboo plants were measured in a single pot, and three pots were measured per treatment. The photosynthesis rate (Pn, µmol CO m 2 s 1), transpiration rate (Tr, mmol H O m 2 s 1), 2• − − 2 • − − water use efficiency (WUE, µmol CO mmol 1 H O), CO of intercellular space (Ci, µL L 1), and 2• − 2 2 • − stomatal conductance (Gs, mmol m 2 s 1) of the bamboo functional leaves were measured using a • − − GFS-3000 (WALZ, Effeltrich, Germany). During the measurement, the environmental condition was set at a sunlight intensity of 1300 µmol m 2 s 1, ambient CO concentration of 400 20 µmol m 2 s 1, • − • − 2 ± • − • − and leaf temperature of 25 1 C[32]. ± ◦ After three days of low-temperature incubations at 5, 0, and 5 C, the SOD, POD, CAT, MDA, − ◦ and cell membrane permeability (CMP) activities of the leaves were measured by the nitroblue Forests 2019, 10, 405 4 of 13 tetrazolium reduction [33], guaiacol colorimetric [34], ultraviolet absorption [35], thiobarbituric acid [36], and electric conductivity methods, respectively [37].

2.5. Observation of Bamboo Leaf Chloroplast Ultrastructure Mature bamboo leaves of the T0 and T4 treatments were collected from the 5 C culture boxes − ◦ and cut into small pieces (1 mm 1 mm). The T0 and T4 treatments were chosen, because T0 had × no Si amendment, and T4 showed the highest leaf biomass among all treatments. We hypothesized that T4 could exhibit a better visual expression than the other treatments. The chosen section was located in the middle of the leaf, at 1 cm from the main vein. Small pieces of these leaf materials were 1 immersed in 4% (m m− ) glutaraldehyde overnight at 4 ◦C, thoroughly rinsed with 0.1 M phosphate buﬀer (pH 7.0), and ﬁxated with 1% (m m 1) osmium acid for 14 h at 4 C. Then, the samples were − − ◦ sequentially immersed in 50% alcohol for 15 min, 70% alcohol for 15 min, 80% alcohol for 15 min (2 times), 90% alcohol for 15 min (2 times), 95% alcohol for 15 min (2 times), 100% alcohol for 10 min (2 times), and 100% acetone for 10 min (2 times). Finally, the samples were embedded in molds with epoxy resin (Epson 812) at 60 ◦C for 24 h. Ultrathin sections were stained by uranyl acetate and lead citrate and observed under a microscope (XSP-8CA, Shanghai, China). Cell photomicrographs were taken by transmission electron microscopy (JEM-1200EX, Tokyo, Japan) [38].

2.6. Data Analysis The study was carried out in a completely randomized design. The SOD, POD, and CAT activity, CMP and MDA concentration, photosynthesis parameters (photosynthesis rate, water use efficiency, CO2 of intercellular space, and stomatal conductance) in the plant leaves, biomass, and root-to-canopy ratio were examined statistically by an analysis of the variance and means of three replicates were subjected to Duncan’s test at a 5% probability level using IBM SPSS Statistics 20.0 (SPSS Inc., Chicago, IL, USA). Furthermore, a multiple regression analysis was adopted. The relationship between the Si content in the plant and the amended rate can be described by the following equation:

Y = ax2 + bx + c (2) where Y is the Si content in bamboo, x is the Si content amended in the pot, and a, b, and c are the parameters for the equation. The relationship between the Si amendment and enzyme content under 5 C treatment could − ◦ also be described by Equation (2), where Y is the SOD, POD, and CAT activity and MDA concentration in the bamboo leaves, and x is the Si content amended in the pot. Correlation coeﬃcients and multiple regression coeﬃcients were calculated using Microsoft Excel.

3. Results

3.1. Bamboo Biomass Change with Si Amendment The bamboo biomass increased significantly when the Si amendment rate was above T2, after six months of incubation (Figure1)( P = 0.002). However, no significant difference was found in the T2, T4, and T8 treatments (P > 0.05). The high rate of Si amendment (T8) led to a slight decline in biomass, compared to T4. Similarly, the root-to-canopy ratio showed the same trend as the biomass ratio increased. T4 had the highest biomass increase and root-to-canopy ratio among the treatments. These results indicated that Si amendment improves bamboo growth under low temperatures. Forests 2019, 10, 405 5 of 13 Forests 2019, 10, x FOR PEER 5 of 14

Figure 1. Bamboo biomass change after Si amendment. Values followed by the same letter(s) are not significantlyFigure 1. Bamboo different biomass at P < change0.05, according after Si amendment. to Duncan’s multipleValues followed range tests. by the The same measurements letter(s) are were not takensignificantly after six different months ofat SiP application< 0.05, according at room to temperature. Duncan’s multiple range tests. The measurements were taken after six months of Si application at room temperature. 3.2. Si Content in Bamboo Plants 3.2. SiThe Content Si contents in Bamboo in the Plants leaf, stem, rhizome, and root of the bamboos increased with the increase of the amendment rate (Table1). In the leaves, the highest Si content was 68.95 mg kg 1, which occurred The Si contents in the leaf, stem, rhizome, and root of the bamboos increased− with the increase in T4. In the stem, rhizome, and root, the highest Si contents were all observed in T8. Relatively, the Si of the amendment rate (Table 1). In the leaves, the highest Si content was 68.95 mg kg−1, which content in the leaf was higher than in the stem, rhizome, or root. The Si content in the bamboo plant occurred in T4. In the stem, rhizome, and root, the highest Si contents were all observed in T8. body can be arranged as follows: leaf > stem > rhizome > root. The results also showed that the Relatively, the Si content in the leaf was higher than in the stem, rhizome, or root. The Si content in total Si content in the bamboo was closely related to the Si amendment rate. Table2 shows that the the bamboo plant body can be arranged as follows: leaf > stem > rhizome > root. The results also Si content in all bamboo organs could be well fitted by the equation, and the equations for the leaf, showed that the total Si content in the bamboo was closely related to the Si amendment rate. Table 2 stem, rhizome, and root were y = 1.2901x2 + 15.311x + 26.607, y = 0.3743x2 + 4.5539x + 10.923, shows that the Si content in all −bamboo organs could be well fitted− by the equation, and the y = 0.1309x2 + 1.5841x + 3.2716, and 0.1003x2 + 1.5574x + 3.1215, respectively. All R2 values were equations− for the leaf, stem, rhizome, −and root were y = −1.2901x2 + 15.311x + 26.607, y = −0.3743x2 + above 0.95. The maximum values of the leaf, stem, rhizome, and root biomass were obtained at 5.93, 4.5539x + 10.923, y = −0.1309x2 + 1.5841x + 3.2716, and −0.1003x2 + 1.5574x + 3.1215, respectively. All R2 6.08, 6.05, and 7.76 mg kg 1, respectively. This result suggests that bamboo had a maximal Si uptake, values were above 0.95. −The maximum values of the leaf, stem, rhizome, and root biomass were regardless of the Si amendment rate. obtained at 5.93, 6.08, 6.05, and 7.76 mg kg−1, respectively. This result suggests that bamboo had a maximalTable Si 1. uptake,Si content regardless in bamboo of parts the withSi amendment various Si treatments rate. (mg kg 1). Data are the means SD of − ± three replicates. Different letters in the same column indicate significant differences, based on Duncan’s −1 multipleTable 1. rangeSi content tests in at bamboo the 0.05 level.parts wi Theth measurementsvarious Si treatments were taken (mg afterkg ). six Data months are the of means Si application ± SD of atthree room replicates. temperature. Different letters in the same column indicate significant differences, based on Duncan’s multiple range tests at the 0.05 level. The measurements were taken after six months of Si applicationTreatment at room temperature. Leaf Stem Rhizome Root T0 27.77 4.70 a1 11.18 1.53 a 3.50 0.72 a 3.07 0.99 a Treatment Leaf ± Stem± ± Rhizome ± Root T0.5 31.93 3.68 a 11.35 1.40 a 3.31 0.81 a 3.69 1.18 a ± ± ± ± T0 T1 27.77 ± 4.70 43.19 a1 3.25 b 11.18 16.71 ± 1.534.37 a ab 4.82 3.500.95 ± a0.72 a 5.33 0.71 3.07b ± 0.99 a ± ± ± ± T2 48.88 2.13 b 18.88 3.69 b 5.64 1.10 ab 6.14 0.74 bc T0.5 31.93 ± 3.68 a± 11.35 ± ±1.40 a ± 3.31 ± 0.81 a ± 3.69 ± 1.18 a T4 68.95 10.11 c 22.60 3.71 c 7.92 2.51 bc 7.16 1.11 c T1 43.19 ± 3.25 b± 16.71 ± 4.37± ab ± 4.82 ± 0.95 a ± 5.33 ± 0.71 b T8 66.26 9.20 c 23.50 5.57 c 9.13 3.25 c 7.63 1.19 c T2 48.88 ± 2.13 b ± 18.88 ± ±3.69 b 5.64± ± 1.10 ab ± 6.14 ± 0.74 bc TableT4 2. Parameters 68.95 for ± 10.11 the equation c for the 22.60 Si content ± 3.71 inc the bamboo 7.92 plant ± 2.51 and bc the Si-amended 7.16 ± plant. 1.11 c SignificantT8 differences 66.26 were± 9.20 based c on Duncan’s 23.50 multiple± 5.57 c range tests 9.13 at the ± 3.25 0.05 c level. 7.63 ± 1.19 c

a b c R2 P

Leaf 1.290 15.31 26.61 0.9828 0.003 − Stem 0.3743 4.554 10.92 0.9566 0.009 − Rhizome 0.1003 1.557 3.122 0.9789 0.003 − Root 0.1309 1.584 3.272 0.9585 0.008 −

Forests 2019, 10, 405 6 of 13

3.3. Photosynthesis Parameters As shown in Table3, with Si amendment, the photosynthesis rate (P n) of the bamboo leaves increased 2 1 2 1 linearly, from 7.03 µmol CO2 m− s− to 11.89 µmol CO2 m− s− , and Pn increased significantly • 1 • when the Si amendment rate was above 4.0 g kg− (P = 0.008). However, the bamboo transpiration rate 2 1 2 1 (Tr) was significantly reduced, from 3.74 mmol H2O m− s− to 1.93 mmol H2O m− s− (P = 0.001). 1 • 1 • The WUE increased from 2.80 µmol CO2 mmol− H2O to 7.39 µmol CO2 mmol− H2O, and it increased • 1 • significantly when the Si amendment rate was above 4.0 g kg− (P = 0.01). The CO2 concentration of the intercellular space and stomatal conductance showed no significant difference with all the Si amending treatments (P > 0.05).

Table 3. Photosynthesis parameters of the bamboo leaf with various treatments. Data are the means SD of three replicates. Different letters in the same column indicate significant differences, ± based on Duncan’s multiple range tests at the 0.05 level. The measurements were taken after six months

of Si application at approximately 25 ◦C.

Water Use Eﬃciency CO of Intercellular Stomatal Photosynthesis Rate Transpiration Rate 2 (WUE) Space Conductance Treatment (Pn) (Tr) (µmol CO mmol 1 (G ) µ 2 1 2 1 2 − (Ci) s ( mol CO2 m− s− ) (mmol H2O m− s− ) • 1 2 1 • • H2O) (µLL ) (mmol m s ) − • − − T0 7.03 2.69 a 3.74 1.26 a 2.80 1.83 a 224.33 45.89 a 109.0 14.91 a ± ± ± ± ± T0.5 7.01 0.65 a 2.64 0.89 b 2.38 0.58 ab 240.62 40.19 a 99.4 28.6 a ± ± ± ± ± T1 8.11 3.97 ab 2.5 0.32 b 3.84 1.58 ab 241.12 45.21 a 97.63 12.83 a ± ± ± ± ± T2 9.57 2.6 abc 2.58 0.89 b 3.94 1.17 ab 240.19 47.57 a 111.07 30.65 a ± ± ± ± ± T4 10.11 2.55 bc 2.27 0.6 b 4.55 1.12 b 228.47 19.73 a 103.96 17.01 a ± ± ± ± ± T8 11.89 1.63 c 1.96 0.75 b 7.39 3.79 c 230.99 37.23 a 118.7 38.06 a ± ± ± ± ±

3.4. Physiological Indicators Treated with Low Temperature Figure2 shows that the SOD activity was significantly higher at 0 C than at 5 C and 5 C ◦ ◦ − ◦ (P = 0.001). With Si amendment, the SOD activity increased with all temperature treatments and was in the order of T8 > T4 > T2 > T1 > T0.5. As shown in Figure3, the low temperature of 5 C significantly − ◦ reduced the POD activity of the bamboo leaves (P = 0.001). The Si amendment increased the POD activity of leaves at various temperatures, and no significant difference was found at 5 ◦C(P > 0.05). At 0 ◦C and 5 ◦C, the POD significantly increased with the increasing rate (P = 0.007, P = 0.002). − 1 Similarly, with no more than 8 g kg− application rates, the SOD and CAT activity of the bamboo leaves increased with the increasing Si amendment (Table4). The highest CAT activity was recorded at 5 C − ◦ with all Si treatments (Figure4). Unlike the SOD and CAT, the Si amendment decreased the MDA concentration (Figure5). The highest MDA concentration was recorded at 5 C. At 0 C and 5 C, − ◦ ◦ − ◦ the MDA significantly decreased with the increasing rate (P = 0.01, P = 0.002) (Table4). The CMP of the bamboo leaves increased with the decreasing temperature (Figure6). The Si amendment could reduce the CMP at various temperatures, and the CMP decreased significantly when the Si amendment rate was above 2.0 g kg 1 at 5 C (Figure1)( P = 0.001). − − ◦ Table 4. Parameters for the equation for the Si amendment and enzyme content at 5 C. Significant − ◦ differences were based on Duncan’s multiple range tests at the 0.05 level. SOD, superoxide dismutase; POD, peroxidase; CAT, catalase; MDA, malondialdehyde.

a b c R2 P SOD 1.783 23.266 633.6 0.89389 0.016 − POD 0.7196 9.935 112.1 0.96943 0.002 − CAT 0.3301 6.390 27.49 0.98354 9.82 10 4 − × − MDA 0.05001 1.202 27.95 0.97371 0.002 − Forests 2019, 10, x FOR PEER 7 of 14

ForestsTable 2019, 104., Parametersx FOR PEER for the equation for the Si amendment and enzyme content at −5 °C. Significant7 of 14 differences were based on Duncan’s multiple range tests at the 0.05 level. SOD, superoxide Table 4. Parameters for the equation for the Si amendment and enzyme content at −5 °C. Significant dismutase; POD, peroxidase; CAT, catalase; MDA, malondialdehyde. differences were based on Duncan’s multiple range tests at the 0.05 level. SOD, superoxide dismutase; POD, peroxidase; CAT, catalase; MDA, malondialdehyde. a b c R2 P SOD −1.783 23.266 633.6 0.89389 0.016 a b c R2 P POD −0.7196 9.935 112.1 0.96943 0.002 SOD −1.783 23.266 633.6 0.89389 0.016 CAT −0.3301 6.390 27.49 0.98354 9.82 × 10−4 POD −0.7196 9.935 112.1 0.96943 0.002 MDA 0.05001 −1.202 27.95 0.97371 0.002 Forests CAT2019, 10 , 405 −0.3301 6.390 27.49 0.98354 9.82 × 10−4 7 of 13 MDA 0.05001 −1.202 27.95 0.97371 0.002

Figure 2. Effect of Si amendment on the SOD activity of leaves at various temperatures. Data are the meansFigure 2.± ESDffect of of three Si amendment replicates. on Different the SOD activityletters at of the leaves same at various temperature temperatures. indicateData significant are the Figuremeans 2. SDEffect ofthree of Si replicates.amendment Di onfferent the SOD letters activity at the same of leaves temperature at various indicate temperatures. significant Data diff erences,are the differences,± based on Duncan’s multiple range tests at the 0.05 level (the same hereinafter). SOD, meansbased on± SD Duncan’s of three multiple replicates. range Different tests at the letters 0.05 levelat the (the same same temperature hereinafter). indicate SOD, superoxidesignificant superoxide dismutase. SOD activities were measured after three days of low-temperature differences,dismutase. SODbased activities on Duncan’s were measured multiple afterrange three tests days at the of low-temperature0.05 level (the same incubations hereinafter). at 5 C, SOD, 0 C, incubations at 5 °C, 0 °C, and −5 °C (the same hereinafter). ◦ ◦ superoxideand 5 C (thedismutase. same hereinafter). SOD activities were measured after three days of low-temperature − ◦ incubations at 5 °C, 0 °C, and −5 °C (the same hereinafter).

Forests 2019, 10, x FOR PEER 8 of 14 Figure 3. Eﬀect of Si amendment on the POD activity of leaves at various temperatures. POD, peroxidase. Figure 3. Effect of Si amendment on the POD activity of leaves at various temperatures. POD, peroxidase. Figure 3. Effect of Si amendment on the POD activity of leaves at various temperatures. POD, peroxidase.

Figure 4. Eﬀect of Si amendment on the CAT activity of leaves at various temperatures. CAT, catalase. Figure 4. Effect of Si amendment on the CAT activity of leaves at various temperatures. CAT, catalase.

Figure 5. Effect of Si amendment on the MDA concentration of leaves at various temperatures. MDA, malondialdehyde.

Figure 6. Effect of Si amendment on the cell membrane permeability of leaves at various temperatures.

Forests 2019, 10, x FOR PEER 8 of 14

Figure 4. Effect of Si amendment on the CAT activity of leaves at various temperatures. CAT, catalase. Figure 4. Effect of Si amendment on the CAT activity of leaves at various temperatures. CAT, Forests 2019 10 catalase., , 405 8 of 13

Figure 5. Effect of Si amendment on the MDA concentration of leaves at various temperatures. MDA, malondialdehyde.Figure 5. Eﬀect of Si amendment on the MDA concentration of leaves at various temperatures. FigureMDA, 5. malondialdehyde. Effect of Si amendment on the MDA concentration of leaves at various temperatures. MDA, malondialdehyde.

Figure 6. Effect of Si amendment on the cell membrane permeability of leaves at various temperatures. Figure 6. Effect of Si amendment on the cell membrane permeability of leaves at various 3.5. Etemperatures.ffect of Si Amendment on Bamboo Leaf Chloroplast Ultrastructure Figure 6. Effect of Si amendment on the cell membrane permeability of leaves at various Figure7 shows that the low temperature of 5 C exerted a significant effect on the bamboo temperatures. − ◦ leaf chloroplast. The chloroplast swelled to a circular shape and separated with the cell membrane. Even worse, the chloroplast membrane ruptured, and the grana disintegrated, while some dissolved. The osmiophillic number increased, and some small vesicles were found in the cell matrix (Figure7A,B). The chloroplast showed an intact ultrastructure with Si treatment (Figure7C,D) and came in close contact with the cell membrane. The membrane of the chloroplast was distinct and full. The application of Si prevented low-temperature stress in the bamboo leaf chloroplast and decreased the degree of damage affecting bamboo growth. Forests 2019, 10, x FOR PEER 9 of 14

3.5. Effect of Si Amendment on Bamboo Leaf Chloroplast Ultrastructure Figure 7 shows that the low temperature of −5 °C exerted a significant effect on the bamboo leaf chloroplast. The chloroplast swelled to a circular shape and separated with the cell membrane. Even worse, the chloroplast membrane ruptured, and the grana disintegrated, while some dissolved. The osmiophillic number increased, and some small vesicles were found in the cell matrix (Figure 7A,B). The chloroplast showed an intact ultrastructure with Si treatment (Figure 7C,D) and came in close contact with the cell membrane. The membrane of the chloroplast was distinct and full. The Forestsapplication2019, 10 ,of 405 Si prevented low-temperature stress in the bamboo leaf chloroplast and decreased9 of the 13 degree of damage affecting bamboo growth.

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C Sg D G G CH Os N G V

G CW

° Figure 7. EffectEffect of of Si Si amendment amendment on on the the chloropl chloroplastast ultrastructure of bamboo leaves at −5 CC( (A) × × × − ◦ 15,000 ,, chloroplastchloroplast of of control; control; (B) ( 40,000B) 40,000, chloroplast, chloroplast of control; of control; (C) 15,000 (C) ,15,000 chloroplast, chloroplast of Si treatment; of Si × × × × (treatment;D) 40,000 (,D chloroplast) 40,000 , chloroplast of Si treatment). of Si Os:treatment). Osmiophile Os: Osmiophile globule; Cw: globule; cell wall; Cw: V: cell vacuole; wall; Ch:V: × chloroplast;vacuole; Ch: G: chloroplast; Granum; N: G: nucleus; Granum; Sg: N: starch nucleus; grain. Sg: The starch ultrastructure grain. The of bamboosultrastructure leaves of (T0 bamboos and T4) ° wereleaves observed (T0 and afterT4) were three observed days of after5 C temperaturethree days of incubations. −5 C temperature incubations. − ◦ 4. Discussion Temperature determinesdetermines the the distribution distribution of bambooof bamboo [28,39 [28,39,40],,40], and P. praecoxand P. ispraecox usually is subjected usually tosubjected low temperatures. to low temperatures. Thus, Si amendment Thus, Si amendment has been consideredhas been consi onedered of the one best of methods the best methods to mitigate to low-temperaturemitigate low-temperature stress in P.stress praecox in P., because praecox, Si because is beneficial Si is beneficial for bamboo for growth bamboo [41 growth,42]. [41,42]. The results of this studystudy indicate that Si amendmentamendment significantlysignificantly improves the bamboo root-to-canopy ratioratio ((PP == 5 × 10−44),), which which indicate indicate that that the the growth growth of of underground underground organs organs increased, × − such thatthat bamboobamboo rootsroots easily easily absorbed absorbed nutrients nutrients and and water water from from the the soil. soil. Moreover, Moreover, Si increased Si increased the totalthe total biomass biomass of bamboo of bamboo and and especially especially accumulated accumulated in bamboo in bamboo leaves, leaves, which which was was beneficial beneficial for thefor bamboothe bamboo growth growth and enhancedand enhanced the cold the tolerance cold toleranc of bamboo.e of bamboo. In our findings, In our findings, Si significantly Si significantly increased theincreased photosynthesis the photosynthesis rate of bamboo rate of leaves bamboo (P = leaves0.008) ( andP = the0.008) ultrastructures and the ultrastructures of the chloroplasts, of the whichchloroplasts, was consistent which was with consistent Detmann etwith al. [ 43Detmann]. Moreover, et al. Si improved[43]. Moreover, the leaf Si thickness improved and the surface leaf areathickness of chloroplasts. and surface In area addition, of chloroplasts. Si improved In theadditi erectnesson, Si improved of leaves, the so aserectness to indirectly of leaves, improve so as the to whole-plantindirectly improve photosynthesis the whole-plant [44]. The WUEphotosynthesis increased significantly[44]. The WUE with increased Si amendment significantly (P = 0.01) with due toSi increasedamendment photosynthesis (P = 0.01) anddue reducedto increased water Tr.photosynthes The stomatalis conductanceand reduced and water mesophyll Tr. The conductance stomatal wereconductance limitations and to mesophyll the photosynthetic conductance capacity were oflimitati leavesons [45 to], butthe therephotosynthetic was no significant capacity di offf erenceleaves of[45], stomatal but there conductance was no insignificant all the Si amendingdifference treatmentsof stomatal (P >conductance0.05), which in suggested all the thatSi amending stomatal produce posed a less significant limitation on photosynthesis. However, the high application rate of Si fertilization may have adverse effects on bamboo growth. Active and passive Si uptake both existed in plants, and in the passive process, the supply of silicon in the bamboo depends on the availability of Si(OH)4 in the soil of their growth area and the rates of water uptake and evaporation [42]. When too much silicon accumulated in the bamboo, the transpiration rate significantly decreased (P = 0.001), and the passive process decreased, which caused the decrease in biomass. Thus, Si fertilization is an effective measure for improving the tolerance of bamboos to cold stress, and a Si fertilization rate of 1 2.0~8.0 g kg− is recommended for bamboo growth. Forests 2019, 10, 405 10 of 13

The increased growth was closely related to the stimulation in the enzymatic antioxidant system under cold stress. ROS, including superoxide (O ), H O , and hydroxyl radicals (OH ), 2•− 2 2 • were generated in the plants due to environmental stresses [46,47]. Chloroplasts are the major sites for ROS generation, and oxygen generated in the chloroplasts during photosynthesis can accept electrons passing through the photosystems, thus forming O [48]. When plants were exposed to cold stress 2•− factors, an excessive accumulation of ROS could result in oxidative damage to plant tissues [49–51]. Moradtalab et al. [52] suggested that the major effect of Si on improving the cold tolerance of plants is the mitigation of oxidative stress. Therefore, cold stress resistance is associated with an enhanced antioxidative defense system, which includes antioxidant compounds and several antioxidative enzymes. In our study, Si application stimulated the SOD, POD, and CAT activity of bamboo leaves at various temperatures. Moreover, the content of SOD, POD, and CAT activity increased with the increase of the Si application rate. SOD is the major O scavenger, and its enzymatic action results 2•− in H2O2 and O2 formation. Since H2O2 can diffuse directly across the membrane, H2O2 produced in chloroplasts, mitochondria, and other organelles can also diffuse into the peroxisomes and be scavenged by CAT [53]. The lowest POD content was observed at 5 C. A low content of H O in − ◦ 2 2 plant cells is removed by POD. When the H2O2 content is very high, CAT is mainly responsible for its removal [54]. Temperature stress results in an increase in the MDA content [55]. Therefore, a low temperature of 5 C increased the MDA content of bamboo leaves, and the decreased MDA and CMP − ◦ content indicated that the stability of the cell membrane was improved, the CMP was decreased, and the membrane lipid peroxidation was alleviated, induced by the low temperature, which enhanced the cold tolerance of the bamboos. Plants have developed an antioxidant system, including CAT, guaiacol peroxidase (GPX), glutathione peroxidase (GSH-Px), and the ascorbate–glutathione cycle, to scavenge ROS. The ascorbate–glutathione cycle, including glutathione (GSH), ascorbate (AsA), and related enzymes, such as glutathione reductase (GR) and dehydroascorbate reductase (DHAR), is an important way to scavenge the toxic products in chloroplasts and other non-photosynthetic tissues [56]. Our study did not examine the dynamic changes of GR and DHAR, but showed that the antioxidant enzymes of SOD, POD, and CAT were stimulated by Si addition under cold conditions. In addition, under a low temperature, the morphological structures of organelles change, along with the damaged chloroplasts and starch granules [57]. In our study, the shape of chloroplasts was improved by Si amendment, and the chloroplast membranes were stabilized. Our results supported previous studies that found that, under cold stress, Si amendment enhanced oxidative defense systems and the chlorophyll content, and reduced the absolute electrolyte leakage quantity of corn plantlets [21,22,58,59]. These results indicate that Si enhances the resistance of plants to cold stress. In addition, further field studies should be conducted to test the effects of silicon in improving bamboo cold tolerance. Our low temperature treatment was not conducted for as long as the field situation required, and our study of the mechanism of Si-mediated cold stress needs further development. The mechanism of Si in improving bamboo cold tolerance was as follows: (1) Si improves the bamboo biomass, the Si content in bamboo leaves, the chlorophyll content, and the ultrastructure of chloroplast, which was beneficial in increasing the photosynthesis rate. (2) Antioxidant systems are stimulated by Si, and play an important role in alleviating the peroxidation damage induced by cold stress.

5. Conclusions Our results show that Si application improved the growth of P. praecox and increased the biomass, root-to-canopy ratio, and Pn of bamboo leaves. The method of soil Si amendment was effective in alleviating cold stress in P. praecox. It resulted in an improved chloroplast and cell structure, increased CAT, POD, and SOD activities, and decreased MDA and CMP levels. These findings suggest that Si is 1 beneficial for bamboo growth, although a high rate of application (8 g kg− ) may have adverse effects 1 on bamboo growth, so we recommended a Si fertilization rate range of 2.0~8.0 g kg− , and Si fertilizer enhances the tolerance of bamboo plants to cold stress. Accordingly, Si fertilization is recommended Forests 2019, 10, 405 11 of 13 for bamboos under cold conditions. Furthermore, more field studies need to be conducted to test the practical application of these findings.

Author Contributions: Conceptualization, Z.Z.Q., S.Y.Z., Q.L., and R.Y.G.; Methodology, Z.Z.Q., S.Y.Z., Q.L., and R.Y.G.; Validation, Z.Z.Q., S.Y.Z., Q.L., and R.Y.G.; Formal Analysis, S.Y.Z.; Resources, S.Y.Z. and R.Y.G.; Visualization, Z.Z.Q., S.Y.Z., Q.L., and R.Y.G.; Investigation, Z.Z.Q. and Q.L.; Resources, S.Y.Z. and R.Y.G.; Software, Z.Z.Q. and Q.L.; Data Curation, Z.Z.Q. and Q.L.; Writing—Original Draft Preparation, Z.Z.Q. and S.Y.Z.; Writing—Review and Editing, Z.Z.Q. and S.Y.Z.; Supervision, S.Y.Z. and R.Y.G.; Project Administration, S.Y.Z.; Funding Acquisition, S.Y.Z. and R.Y.G. Funding: The authors are grateful for financial support from the National Natural Science Foundation of China (41671296), National Key R & D Project of China (2016FYE0112700), and Science and Technology Department of Zhejiang Province of China (2017C02016). Acknowledgments: We are grateful for experimental support from Yuhe Zhang and Xin Wang. Conflicts of Interest: The authors declare no conflict of interest.

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